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We investigate the hypothesis that, in Escherichia coli, while the concentration of RNA polymerases differs in different growth conditions, the fraction of RNA polymerases free for transcription remains approximately constant within a certain range of these conditions. After establishing this, we apply a standard model-fitting procedure to fully characterize the in vivo kinetics of the rate-limiting steps in transcription initiation of the Plac/ara-1 promoter from distributions of intervals between transcription events in cells with different RNA polymerase concentrations. We find that, under full induction, the closed complex lasts ∼788 s while subsequent steps last ∼193 s, on average. We then establish that the closed complex formation usually occurs multiple times prior to each successful initiation event. Furthermore, the promoter intermittently switches to an inactive state that, on average, lasts ∼87 s. This is shown to arise from the intermittent repression of the promoter by LacI. The methods employed here should be of use to resolve the rate-limiting steps governing the in vivo dynamics of initiation of prokaryotic promoters, similar to established steady-state assays to resolve the in vitro dynamics.

Arylhydrazones of active methylene compounds (AHAMCs) are potent chemotherapy agents for the cancer treatment. AHAMCs enhance the apoptotic cell death and antiproliferation properties in cancer cells. In this study, a series of AHAMCs, 13 compounds, was assayed for cytotoxicity, apoptosis, externalization of phosphatidylserine, heterogeneity and cellular calcium level changes. The in vitro cytotoxicity study against HEK293T cells suggests that AHAMCs have significant cytotoxic effect over the concentrations. Top 5 compounds, 5-(2-(2-hydroxyphenyl) hydrazono)pyrimidine-2,4,6(1H,3H,5H)-trione (5), 4-hydroxy-5-(2-(2,4,6-trioxo-tetrahydro-pyrimidin-5(6H) ylidene)hydrazinyl)benzene-1,3-disulfonic acid (6), 5-chloro-3-(2-(4,4-dimethyl-2,6-dioxocyclohexylidene)hydrazinyl)-2-hydroxybenzenesulfonic acid (8), 5-(2-(4,4-dimethyl-2,6-dioxocyclohexylidene)hydrazinyl)-4-hydroxybenzene-1,3-disulfonic acid (9) and 2-(2-sulfophenylhydrazo)malononitrile (10) were chosen for the pharmacodynamics study. Among these, compound 5 exhibited the better cytotoxic effect with the IC50 of 50.86 ± 2.5 mM. DNA cleavage study revealed that 5 induces cell death through apoptosis and shows more effects after 24 and/or 48 h. Independent validation of apoptosis by following the externalization of phosphatidylserine using Annexin-V is also in agreement with the potential activity of 5. Single cell image analysis of Annexin-V bound cells confirms the presence of mixture of early, mid and late apoptotic cells in the population of the cells treated with 5 and a decreased trend in cell-to-cell variation over the phase was also identified. Additionally, intracellular calcium level measurements identified the Ca2+ up-regulation in compound treated cells. A brief inspection of the effect of the compound 5 against multiple human brain astrocytoma cells showed a better cell growth inhibitory effect at micro molar level. These systematic studies provide insights in the development of novel AHAMACs compounds as potential cell growth inhibitors for cancer treatment. publishedVersion

Phenolic compounds are known for their cytotoxic properties against cancer cells despite their still unclear general mechanism of action. Herein is reported the evaluation of the cytotoxic effects of on human osteosarcoma cells of nine phenol derivatives against osteosarcoma cells, and some insights on their mechanism. The cytotoxicity was characterized by cell viability, scratch assay, cellular DNA content measurement, Annexin V apoptosis, mitochondrial calcium and caspase 3/7 assays. The study shows that out of the nine compounds used in this study, a tetrahydroquinoline derivative, 2-((1,2,3,4-tetrahydroquinolin-1-yl)(4-methoxyphenyl)methyl)phenol, was found to exhibit strong inhibitory response with IC50 of 50.5 ± 3.8 μM, and therefore can be a potential chemotherapeutic agent. Further experiments revealed that this compound induces cell death by apoptosis and also act as a migration inhibitor. Analysis of the mitochondrial calcium following treatment with the compound on U2OS cells showed a significant reduction in the level of mitochondrial calcium concentration suggesting a mitochondrial calcium-independent mechanism in triggering apoptosis. Treatment of HEK293 cells with the compound confirmed the cytotoxic effects of the compound, however, an increase in the level of mitochondrial calcium was observed. Moreover, the caspase 3/7 mediated cell death was also observed in both cell types. Overall, the study suggests that the derivatives of this compound can be used for development of new therapeutics for osteosarcoma and other cancers.

Development of novel anticancer drugs is inevitable to improve treatment of cancers. In this study, novel derivatives of indoline and morpholine were synthesized and tested for their cytotoxic effects on osteosarcoma and Human Embryonic Kidney cells. To characterize cytotoxicity and the mechanism of cell death, we used cytotoxicity, migration, apoptosis markers and mitochondrial calcium assays. Among the compounds tested, the indoline derivatives, generally, produced a higher cytotoxic effect compared to the morpholine derivatives, in osteosarcoma cells. Specifically, new indoline derivative N-(2-hydroxy-5-nitrophenyl(4'-methylphenyl)methyl)indoline exhibited effective cytotoxic activity, with an IC50 of ∼74 μM. The same molecule induced cell death by apoptosis and inhibited migration of the cells. Further, analysis of mitochondrial calcium levels revealed the existence of calcium dependent cell death mechanisms in different cell types. Therefore, N-(2-hydroxy-5-nitrophenyl(4'-methylphenyl)methyl)indoline can be considered as a potential drug-lead compound towards the discovery of new anti-cancer agents.

Synthetic genetic clocks, such as the Elowitz–Leibler repressilator, will be key regulatory components of future synthetic circuits. We constructed a single-copy repressilator (SCR) by implementing the original repressilator circuit on a single-copy F-plasmid. After verifying its functionality, we studied its behaviour as a function of temperature and compared it with that of the original low-copy-number repressilator (LCR). Namely, we compared the period of oscillations, functionality (the fraction of cells exhibiting oscillations) and robustness to internal fluctuations (the fraction of expected oscillations that would occur). We found that, under optimal temperature conditions, the dynamics of the two systems differs significantly, although qualitatively they respond similarly to temperature changes. Exception to this is in the functionality, in which the SCR is higher at lower temperatures but lower at higher temperatures. Next, by adding IPTG to the medium at low and high concentrations during microscopy sessions, we showed that the functionality of the SCR is more robust to external perturbations, which indicates that the oscillatory behaviour of the LCR can be disrupted by affecting only a few of the copies in a cell. We conclude that the SCR, the first functional, synthetic, single-copy, ring-type genetic clock, is more robust to lower temperatures and to external perturbations than the original LCR. The SCR will be of use in future synthetic circuits, since it complements the array of tasks that the LCR can perform.

Using a single-RNA detection technique in live Escherichia coli cells, we measure, for each cell, the waiting time for the production of the first RNA under the control of PBAD promoter after induction by arabinose, and subsequent intervals between transcription events. We find that the kinetics of the arabinose intake system affect mean and diversity in RNA numbers, long after induction. We observed the same effect on Plac/ara-1 promoter, which is inducible by arabinose or by IPTG. Importantly, the distribution of waiting times of Plac/ara-1 is indistinguishable from that of PBAD, if and only if induced by arabinose alone. Finally, RNA production under the control of PBAD is found to be a sub-Poissonian process. We conclude that inducer-dependent waiting times affect mean and cell-to-cell diversity in RNA numbers long after induction, suggesting that intake mechanisms have non-negligible effects on the phenotypic diversity of cell populations in natural, fluctuating environments.

We studied the behaviour of the repressilator at 28 °C, 30 °C, 33 °C, and 37 °C. From the fluorescence in each cell over time, we determined the period of oscillations, the functionality (fraction of cells exhibiting oscillations) and the robustness (fraction of expected oscillations that occur) of this circuit. We show that the oscillatory dynamics differs with temperature. Functionality is maximized at 30 °C. Robustness decreases beyond 30 °C, as most cells exhibit 'failed' oscillations. These failures cause the distribution of periods to become bimodal, with an 'apparent period' that is minimal at 30 °C, while the true period decreases with increasing temperature. Based on previous studies, we hypothesized that the failures are due to a loss of functionality of one protein of the repressilator, CI. To test this, we studied the kinetics of a genetic switch, formed by the proteins CI and Cro, whose expression is controlled by PRM and PR, respectively. By probing the activity of PRM by in vivo detection of MS2-GFP tagged RNA, we find that, beyond 30 °C, the production of the CI-coding RNA changes from sub-Poissonian to super-Poissonian. Given this, we suggest that the decrease in efficiency of CI as a repressor with temperature hinders the robustness of the repressilator beyond 30 °C. We conclude that the repressilator is sensitive but not robust to temperature. Replacing CI for a less temperature-dependent protein should enhance robustness.

We present a new quantitative method of estimation of fluorescent molecule numbers from time-lapse, single-cell, fluorescence microscopy data. Its main aim is to eradicate backward propagation of noise, which is present in previous methods. The method is first validated using Monte Carlo simulations. These tests show that when the time-lapse data are corrupted with negative noise, the method obtains significantly more precise results than current techniques. The applicability of the method is demonstrated on novel time-lapse, single-cell measurements of fluorescently tagged ribonucleic acid (RNA) molecules. Interestingly, we find that the intervals inferred by the new method have the same mean but reduced variability when compared to the previously existing method, which, in accordance to human observers, is a more accurate estimation.